N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated from the olive trees affected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 µM. Cell surface adhesion was dramatically reduced at 500 µM NAC (−47%), hence, this concentration was selected for investigating the effects of pre-, post- and co-treatments on biofilm physiology and structural development, oxidative homeostasis, and biofilm detachment. Even though 500 µM NAC reduced bacterial attachment to surfaces, compared to the control samples, it promoted Xf-DD biofilm formation by increasing: (i) biofilm biomass by up to 78% in the co-treatment, (ii) matrix polysaccharides production by up to 72% in the pre-treatment, and (iii) reactive oxygen species levels by 3.5-fold in the co-treatment. Xf-DD biofilm detachment without and with NAC was also investigated. The NAC treatment did not increase biofilm detachment, compared to the control samples. All these findings suggested that, at 500 µM, NAC diversified the phenotypes in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm. This study investigated in-vitro the non-lethal effects of  Xylella fastidiosa is a bacterium that infects more than 500 different plants including ornamental plants, landscape trees, wild and crop species [X.-fastidiosa-related epidemic diseases in valuable crops have been well documented [X. fastidiosa in the European Union dated back in October 2013, when the Italian Phytosanitary Authorities reported the presence of this quarantined pathogen in southern Italy’s olive orchards [X. fastidiosa-related disease called olive quick decline syndrome (OQDS), known to cause severe leaf scorth and branch dieback, poses one of the most debilitating threats to the European agriculture and landscape [X. fastidiosa subspecies pauca strain De Donno (Xf-DD) was recovered in 2014 from OQDS-affected olive trees. Its complete genome has been sequenced [ species ,2. Seriocumented . The firorchards . The novandscape . X. fastequenced  and the equenced .X. fastidiosa relies on the obstruction of xylem vessels caused by biofilm formation [The major pathogenic mechanism of ormation ,10. A biormation . Within ormation . While iormation . Xylella-associated diseases nor a strategy to contain its impact. The spread of the disease is minimized by simply removing the infected plants and reducing the vector populations [So far, there has been neither a therapy to cure ulations . Howeverulations ,15, the ulations  and the ulations .X. fastidiosa cells [Other studies have reported some strategies that directly target the pathogen to reduce its spread inside the plant. These strategies are aimed to kill the phytopathogen by using specific phages that are able to lysate sa cells , antibiosa cells ,20, metasa cells ,22, and sa cells ,24,25. N-acetylcysteine (NAC), a thiol-containing compound commonly found in Allium plants, has shown interesting antibiofilm activities in clinical settings [X. fastidiosa strain 9a5c, isolated from citrus variegated chlorosis (CVC) symptomatic sweet orange trees, and other phytopathogenic bacteria with lethal NAC concentrations. In-vitro studies have shown that concentrations of NAC of 1.0, 2.0 and 6.0 mg/mL affect X. fastidiosa strain 9a5c adhesion to glass surfaces and EPS amount, consequently reducing the biofilm biomass [X. fastidiosa strain 9a5c [Among natural bioactive molecules, settings ,27,28. Msettings  and Piccsettings  reported biomass . The redain 9a5c . In-vivoain 9a5c . X. fastidiosa has never been investigated. This is an important aspect to be studied, because for each biocide treatment, the concentration of the biocide is reasonably expected to diminish over time and space, resulting in a non-lethal level away from the application point. The physiological and behavioral responses of X. fastidiosa biofilm at this low concentration may be different from those at biocidal concentrations. Despite the encouraging results, the non-lethal effects of NAC on X. fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm, originally isolated from OQDS-affected olive trees in southern Italy. After the response of Xf-DD to a range of non-lethal concentrations was screened, 500 µM NAC showed the highest effect on reducing the number of cells attached to surfaces, and hence, this concentration was selected for further investigations. Additional biofilm experiments with 500 µM NAC simulated three different treatments: (i) pre-treatment, where planktonic cells were treated with NAC before their attachment to the surface; (ii) post-treatment, where biofilm was grown in the presence of NAC after the attachment phase; and (iii) co-treatment, where planktonic and biofilm cells were exposed to NAC. For the first time, the effects of 500 µM NAC on Xf-DD biofilm were studied, taking into consideration biofilm formation and detachment. To this end, biofilm physiology and structural development, modulation of oxidative homeostasis, and induction of biofilm dislodgment were investigated.With these considerations, the main goal of this study was to investigate in-vitro the non-lethal effects of NAC on Xf-DD strain (CFBP 8402) was originally isolated in 2014 from a OQDS-affected olive tree (Olea Europaea cv Ogliarola) grown in the municipality of Gallipoli  . The strN-acetylcysteine (NAC) was purchased from Sigma-Aldrich . NAC was dissolved in deionized water at room temperature to a concentration of 5000 µM. After adjusting pH to 6.8 with sodium hydroxide (1 M NaOH), the stock solution was sterilized by filtration through a 0.22 µm filter and diluted in PD3 medium to the final concentrations of 1000, 500, 250, 50, 5, 0.5 and 0.05 µM. 600) every 24 h for 7 days using an Infinite F200 PRO microplate reader . The polynomial Gompertz model was used to fit the growth curves and the maximum specific growth (OD600/day) was calculated using the GraphPad Prism software . Experiments were repeated independently three times with at least six technical replicates per time.The ability of 1000 µM of NAC to inhibit the planktonic growth of Xf-DD was investigated as previously reported by Kandel et al. , with soXf-DD colonies grown on PD3 agar plates were scraped and resuspended in 2 mL of PD3 . Sterile polystyrene 96-well plates containing 190 μL PD2 per well supplemented with 0, 1000, 500, 250, 50, 5, 0.5 and 0.05 µM NAC were inoculated with 10 μL of cell suspensions. After 1 day of incubation at 28 °C, the cells suspensions were removed, the wells rinsed 3 times with Milli-Q water and stained with 0.1% crystal violet to quantify the attached cells . The staXf-DD biofilm was grown on a transwell device as previously described by Garuglieri et al.  with somXf-DD biofilm was grown in four different conditions : (1) botg). The supernatant was collected and the protein amount was quantified by Bradford assay [Biofilm biomass was evaluated measuring the amount of cellular protein . Brieflyrd assay  using boSeven-day biofilms grown on the membrane in the transwell devices were transferred in 1 mL of Milli-Q water. Biofilms were dislodged from the membranes and cells were broken as reported in the section ‘Biofilm biomass quantification’. The Live/Dead BacLight viability kit  was used to detect live and dead cells according to the manufacturer’s instructions. The fluorescence intensity was measured using the Infinite 200 PRO Microplate Reader  with excitation at 480 nm and emission at 516 nm for the live green cells, and excitation at 581 nm and emission at 644 nm for the red dead cells. Fluorescence intensity was normalized by the proteins within the biofilm, divided for the area of the membrane, and the means reported. Relative viability within the biofilms was calculated by dividing the fluorescent intensity of normalized live cells by the fluorescent intensity of normalized dead cells in each sample. Experiments were repeated three times with at least six technical replicates per time. Three-dimensional morphology of seven day-biofilms from each treatment were analyzed by CLSM. The biofilms were stained with Sybr green I fluorescent nucleic acid dye  to reveal biofilm cells. The staining was performed by incubating the biofilm with 200 µL of 1:1000 Sybr green I dye solution in PBS at room temperature in the dark for 30 min, and then rinsed with PBS. Membranes without biofilms were also stained in order to exclude any false positive signals. Each biofilm sample was visualized using a Nikon A1 with excitation at 488 nm line, and emission at 500 to 550 nm (green channel). Images were captured with a 100× oil immersion objective and analyzed with the software Fiji .Seven day-biofilms from each treatment were analyzed for their EPS proteins and polysaccharides content. EPS extraction was performed as reported by Villa et al. . The Brag. The supernatant was transferred to another tube, filtered (0.22 µm pores size) and analyzed for the extra-cellular ROS content. The pellet was resuspended in 50 mM PBS, cells were broken by sonication as described previously in the section ‘Biofilm Biomass Quantification’, and the intra-cellular ROS content was quantified. Intra- and extra-cellular oxidative stresses were quantified using the ROS sensitive probe 2,7-dichlorofluorescein-diacetate according to Jakubowski et al. [Seven day-biofilms from each treatment were transferred in 1 mL of 50 mM PBS and the biofilm was detached from the membranes by vortex mixing and sonication as described above in the section ‘Biofilm Biomass Quantification’. The suspension was centrifuged for 20 min at 11,000× i et al. . FluoresMembranes with biofilms grown for 7 days under the different treatments were transferred in petri dishes (ø 60 mm) and incubated with 2 mL of phosphate buffered saline solution  or 500 µM NAC for further 24 h at 28 °C. Subsequently, biofilms on the membranes and those dislodged in the bulk liquid were analyzed for their biomass and the level of ROS as previously described in ‘Biofilm Biomass Quantification’ and ‘Oxidative Stress Assay’. The detachment index was calculated as: (protein amount in the detached biofilm × 100)/(protein amount detached biofilm + protein amount in the biofilm on the membrane). Experiments were repeated independently three times with at least four technical replicates per time.t-test analysis, via a software run in MATLAB environment , were applied to statistically evaluate any statistically significant differences among the samples. ANOVA and Student’s t-test analysis were carried out after verifying data independence (Pearson’s chi-square test), normal distribution  and homogeneity of variance (Bartlett’s test). Tukey’s honestly significant different test (HSD) was used for pairwise comparison to determine the significance of the data. Statistically significant results were depicted by p-values ≤ 0.05.Two-tailed ANOVA and Student’s 600/day) between the NAC-treated samples  and control samples . Therefore, 1000 µM NAC was considered a non-lethal concentration.The effects of 500 µM NAC on Xf-DD biofilm biomass were assessed by quantifying the protein amount within the biofilm. The data showed that 500 µM NAC significantly increased the amount of biofilm proteins in all the treatments, compared to the control a, highliThe Live/Dead viability assay was performed to verify the ability of NAC to affect biofilm viability. Relative viability calculated for each condition revealed no statistically significant differences among samples b.Representative biofilm structures observed under different treatments are presented in Inside the EPS b, a statIn order to study Xf-DD biofilm detachment after NAC treatments, an experiment was carried out in which mature biofilms, grown without and with NAC according to the pre-, post- and co- treatments, were soaked in both PBS and 500 µM NAC. After 24 h the biomass remaining on the membrane surface and those released in the bulk liquid were quantified. The tendency towards biofilm detachment for the control Xf-DD biofilm soaked in PBS was 3-fold higher than that soaked in NAC, suggesting that NAC increased Xf-DD cell-cell adhesion . CompariAfter detachment experiments, the level of ROS was quantified in Xf-DD biomass remained on the membrane, in the biomass detached from the membrane and in the bulk liquid medium. The data revealed that the biomass retained on the membrane a after sEnterococcus faecalis and Staphylococcus aureus, and Gram-negative pathogenic bacteria such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia, as well as some yeasts [X. fastidiosa strain 9a5c isolated from symptomatic sweet orange trees. Indeed, both in-vitro and in-vivo findings indicated that NAC at lethal concentrations can be an effective strategy for attenuating the CVC symptoms in citrus. However, downstream from the treated area, there is likely to be a gradient of the biocide concentration, ranging from the prescribed concentration to virtually zero. The response of the plant pathogens to this low, non-lethal concentration of NAC was the impetus of this study. Many in-vitro studies have demonstrated NAC antibiofilm activity against a variety of Gram-positive, such as e yeasts ,42,43,44e yeasts ,46,47. Te yeasts  to test For the first time, this work investigates the non-lethal effects of 500 µM NAC on Xf-DD biofilm by simulating pre- (NAC/–), post- (–/NAC) and co- (NAC/NAC) treatments in-vitro. The 500 µM NAC concentration was selected after screening the response of Xf-DD to a range of non-lethal NAC concentrations, where an NAC concentration of 500 µM reduced Xf-DD surface adhesion by 47% in microtiter assays. Interestingly, the best anti-adhesion performance of NAC was obtained at the specific threshold level of 500 μM, which did not correspond to the maximum concentration tested. The non-linear response patterns depicted by the anti-adhesion assays followed a parabola-like shape profile resembling a hormetic property, a situation in which the response to a compound varies with the exposure level. A biphasic profile is common in the biofilm world ,49. For While microtiter techniques are more suitable for studying bacterial adherence to a surface, the static nature of these techniques has a tendency towards cell nutrient exhaustion that limits the generation of mature biofilm . Therefo3+, which is reduced to its catalytically-active form Fe2+. Furthermore, NAC can react with free radicals and become a thiyl radical itself [X. fastidiosa, ROS acted as an environmental cue that stimulated biofilm development during the early stage of plant colonization, such that the oxidative imbalance was actually required by X. fastidiosa to achieve maximum xylem colonization [X. fastidiosa biofilm is related to the sensing of a redox imbalance via the oxidative stress regulator OxyR [oxyR mutants were impaired in their ability to attach to surfaces, to aggregate with other cells, and to consequently form biofilm. Thus, biofilm formation by X. fastidiosa might be an adaptive response to the exogenous oxidative stress normally encountered by the bacterium in the xylem vessels [Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa caused by the combination of NAC and serum transferrin. The authors demonstrated that biofilm potentiation is caused by a disturbance of the redox status in the bacterial cells. Eroshenko et al. [S. aureus and E. faecalis biofilms increased when exposed to sub-lethal concentrations of NAC.Moreover, biofilms exposed to NAC were more prone to accumulate both intracellular and extracellular ROS. Although NAC is well known for its antioxidant activity, it can act as a pro-oxidant under specific conditions . NAC genl itself ,53. In Xnization . It has tor OxyR ,55. In f vessels ,55,56,57 vessels ,58,59,60 vessels , where to et al.  also fouKlebsiella pneumoniae biofilm after being exposed to NAC. In addition, Yin et al. [S. aureus. The polysaccharide overproduction in the EPS matrix after NAC exposure could be related to the oxidative imbalance in the treated biofilm. It has been demonstrated that EPS polysaccharides played a critical role in relief of the oxygen stress in several microorganisms such as Pseudomonas putida, Sinorhizobium meliloti and Acinetobacter oleivorans [oxyR mutants negatively regulated the gene expression of exopolysaccharides, making the mutants more prone to oxidative stress. Besides providing protection against ROS and other environmental injuries, the polysaccharide component allows the cells to adhere to each other and to the surfaces [X. fastidiosa, exopolysaccharide production is fundamental to surface attachment, biofilm formation within the xylem vessels, virulence and insect transmission [X. fastidiosa subsp. fastidiosa, Killiny et al. [All NAC-treated samples showed an increase in the EPS polysaccharides/EPS proteins ratios. Indeed, the ratio was shown to increase with the protraction of NAC exposure, with ratios from 1:1 in the control biofilm to 15:1 in the case of NAC/NAC. This result suggests that increased exposure to sub-lethal NAC levels promoted polysaccharide production in the EPS matrix. Further, polysaccharides increase the cohesiveness of the biofilm thereby reducing detachment , as seenn et al.  showed aeivorans ,64,65,66eivorans ,68. Inteeivorans . Burbankeivorans  demonstrsurfaces . In X. fsmission ,72. By ky et al.  demonstry et al. . The impact of NAC treatment on Xf-DD biofilm detachment was also examined. The results indicated that none of the NAC-based treatments increased biofilm detachment, compared to the control samples. Rather, control samples (–/–) that were further exposed to NAC became less prone to biofilm disaggregation and detachment. X. fastidiosa forms a biofilm to partially attenuate its own virulence by limiting its diffusion within the plant [X. fastidiosa biofilm with a high degree of adhesion, which inhibited the acquisition and the transmission of pathogens by sharpshooter vectors as well as the fast colonization of the plant throughout the xylem vessels. Thus, this study provides some important implications on the ecology of Xf-DD biofilm, as well as how this plant pathogen should be treated. Overall, our findings indicated that 500 µM NAC had generated phenotypic diversification in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm. This results are in line with a number of studies reporting how exposure to sub-lethal doses of antibiotics may enhance biofilm formation in a wide range of species ,76. Desphe plant . Newman he plant  observedFuture works will be devoted to deciphering the mechanisms behind the non-lethal effects of NAC on Xf-DD. Further in-planta investigations are necessary to elucidate the effects of non-lethal concentrations of NAC on Xf-DD virulence.


In this work, three-dimensional (3D) colloidal CIS nanoflowers and nanospheres and two-dimensional (2D) nanoplatelets were selectively synthesized by changing the amount of a sulfur precursor (tert-dodecanethiol) serving both as a sulfur source and as a co-ligand. Monodisperse CIS nanoflowers (~15 nm) were formed via the aggregation of smaller CIS nanoparticles when the amount of tert-dodecanethiol used in reaction was low enough, which changed towards the formation of larger (70 nm) CIS nanospheres when it significantly increased. Both of these structures crystallized in a chalcopyrite CIS phase. Using an intermediate amount of tert-dodecanethiol, 2D nanoplatelets were obtained, 90 nm in length, 25 nm in width and the thickness of a few nanometers along the a-axis of the wurtzite CIS phase. Based on a series of experiments which employed mixtures of tert-dodecanethiol and 1-dodecanethiol, a ligand-controlled mechanism is proposed to explain the manifold range of the resulting shapes and crystal phases of CIS nanostructures.Colloidal semiconductor nanostructures have been widely investigated for several applications, which rely not only on their size but also on shape control. CuInS Colloidal semiconductor nanostructures have been extensively investigated due to their size dependent electronic structure and their attractive optical properties ,2. The b2 (often abbreviated as CIS) is a heavy metal-free and earth abundant ternary semiconductor, which has a direct band gap of ~1.5 eV and a Bohr exciton radius of ~4.1 nm [5 cm−1 at 500 nm, [2S NCs can form at the initial stage, and ternary CIS NCs can then be formed via a partial cation exchange. [2S-In2S3 and Cu2S-CIS, were also reported in some cases [tert-dodecanethiol (t-DDT) which played a crucial role in the formation of CIS nanostructures reported here.CuInS ~4.1 nm . It is a 500 nm,  which ma 500 nm,  Colloida 500 nm,  Colloida 500 nm, . Becausexchange.  Based onme cases ,20. We nme cases , and broChemicals: Indium (III) acetate (99.99%), copper (I) iodide (98%), tert-dodecanethiol , 1-dodecanethiol , 1-octadecene , toluene , and ethanol  were purchased from Sigma-Aldrich. Oleylamine  was purchased from Acros.The experiments below were carried out using standard airless techniques: a vacuum/dry Ar gas Schleck line was used for the syntheses.Synthesis of CIS nanoflowers: Indium acetate , copper iodide , OLA (4.0 mL) and ODE (8.0 mL) were added into a three-neck flask which was then connected to a Schleck line. The mixture in the three-neck flask was degassed and heated to 90 °C, and the mixed solution was kept at 90 °C under vacuum for 1 h to remove any water from the system. Then the solution was heated to 180 °C to form a clear solution. At 180 °C, 0.5 mL of t-DDT was injected into the three neck flask. Then the reaction was kept at 180 °C for 1h. The crude CIS nanoflowers product solution was dissolved in toluene, and the CIS nanoflowers were precipitated by adding ethanol and with the aid of centrifugation. All of the CIS nanoflower products were purified by dissolving in toluene and precipitating with ethanol for three cycles, and were finally re-dissolved in toluene.Synthesis of CIS NPLs: The synthetic process was similar to the synthesis of CIS nanoflowers, with the difference being that the injected amount of t-DDT was increased. Thus, 1.0 mL of t-DDT was injected into the three-neck flask at 180 °C with 0.5 mmol of copper precursor and 0.5mmol indium precursor in OLA and ODE. Then, the reaction was kept at 180 °C for 1 h. The crude CIS NPL product solution was dissolved in toluene, and the CIS NPLs, as above, were precipitated by adding ethanol with the aid of centrifugation. Again, all of the CIS NPL products were purified by dissolving in toluene and precipitating with ethanol for three cycles and were finally re-dissolved in toluene.Synthesis of CIS nanospheres: The synthetic process was similar to the synthesis of CIS nanoflowers and NPLs, but in this case the injected amount of t-DDT was increased still further. Here 2.0 mL of t-DDT was injected into the three-neck flask at 180 °C with 0.5 mmol of copper precursor and 0.5 mmol indium precursor in OLA and ODE. Then the reaction was kept at 180 °C for 1 h. The crude CIS nanospheres product solution was dissolved in toluene, and the CIS nanospheres were precipitated by adding ethanol with the aid of centrifugation. All of the CIS nanospheres products were purified by dissolving in toluene and precipitating with ethanol for three cycles, and were finally re-dissolved in toluene.Characterization: Transmission electron microscopy (TEM) was performed using a Philips CM-20 at an accelerating voltage of 200 kV. TEM grids were prepared by depositing one drop of a solution of purified nanoparticles onto an ultrathin carbon-coated grid. High-resolution TEM (HRTEM) was performed using a JEOL 2100F high-resolution transmission electron microscope running at an accelerating voltage of 200 kV with a field-emission gun as an electron source. The grids for HRTEM were prepared by depositing one drop of a solution of purified nanoparticles onto an ultrathin carbon-coated grid. Powder X-ray diffraction (XRD) patterns were obtained using Cu Kα photons from a Bruker D2 Phaser operated at 40 kV and 30 mA. Each sample was deposited as a thin layer on a low background-scattering quartz substrate. Absorption spectroscopy was performed on a Cary 50 ultraviolet–visible spectrophotometer (Varian) using quartz cuvettes. The samples were dissolved in toluene.t-DDT) was injected into the metal precursors solution, and the amount of this reagent largely determined the shape of final CIS NCs, as will be demonstrated below. t-DDT was 0.5 mL; they had monodisperse sizes of around ~15 nm, and were formed from a few smaller (3–5 nm) CIS nanoparticles  and oleylamine (OLA). At 180 °C, the sulfur precursor  spherical particles were formed . In order to further reveal the detailed structure of CIS NCs, HRTEM was employed. Similar to CIS nanoflowers, 3D CIS nanospheres with an average size of around 70 nm were formed by the aggregation of small CIS NCs, as shown in NPLs are an interesting class of 2D nanostructures, which can show a strong quantum size confinement effect in one direction . CdSe NPLs with different numbers of layers have been extensively studied, and have showed strikingly narrow absorption and fluorescence spectra ,7. For tThe thickness is an important parameter for 2D nanostructures, as the quantum size confinement effect can be tuned via the thickness control in such materials. In some CIS NPLs with roughened edges a, severat-DDT. The t-DDT plays a key role in the shape-controlled synthesis of CIS NCs, acting as both a sulfur precursor and a ligand. This reaction component has been used before to synthesize Cu-In-Zn-S nanoflowers [t-DDT was the reason for the aggregation of smaller NCs to form larger scale nanoflowers. They also observed that the use of the less t-DDT led to the formation of larger size Cu-In-Zn-S nanoflowers. Thus, in order to further understand the role of t-DDT in the shape-controlled synthesis of our CIS NCs, some additional ligand-controlled experiments were conducted, using mixtures of 1-DDT and t-DDT. Notably, 1-DDT has a similar, but not branched, structure to t-DDT, and has also been widely used to synthesize dot-shaped CIS NCs, again fulfilling the roles of both a sulfur precursor and a ligand [t-DDT (1.0 mL) was injected into the copper and indium precursor solution instead of 2.0 mL of t-DDT only, the resulting CIS NCs showed dot-shaped habits with sizes of 3.0~4.0 nm  when used alone can also induce the aggregation of CIS NCs to form differently shaped nanostructures. When 0.5 mL of t-DDT was injected into the reaction, it is estimated that ~0.24 mL of the t-DDT acted as a sulfur precursor and the remaining ~0.26 mL of t-DDT acted as a ligand. The 0.26 mL of t-DDT was not enough to cover all of the surface of the CIS NC product, so this role was partially met by the relatively weaker binding OLA which also acted as a ligand for the CIS NCs. The surface of the CIS NCs NCs and some parts of the t-DDT ligand coverage could now aggregate at a high temperature, and as a result CIS nanoflowers could then be formed by NC aggregation via the openings provided by the partial t-DDT binding over a proportion of the surface substituent NCs. The presence of OLA as a co-ligand prevents the formation of larger CIS NCs, such as nanospheres, and only small-sized CIS nanoflowers were obtained. However, when 2.0 mL of t-DDT was injected into the reaction vessel, its overall amount was sufficient to fully meet the role of ligand for the CIS NCs. In this case, all of the produced CIS NCs were sufficiently covered with the t-DDT ligand. Related to our discussion of t-DDT is a stronger ligand than OLA, and at high temperatures CIS NCs could aggregate in a more extensive manner to form larger nanospheres dominated by this particular ligand. The CIS NPLs (synthesized upon injection of 1.0 mL of t-DDT) are the intermediate case between the nanoflowers and nanospheres, where the CIS NCs carry on t-DDT as the main ligand and a lesser proportion of the surface is still occupied by OLA. The t-DDT ligand then binds preferentially at a high energy surface and the OLA on a lower energy surface, leading to a more ordered assembly into NPLs than that which occurs with either an over- or an under-abundance of the thiol ligand.In the same vein as the discussions above, moderating the amount of the t-DDT reactant acting as both a sulfur precursor and a ligand. CIS nanoflowers were formed by the aggregation of a few small (3–5 nm) CIS NCs, and possessed the chalcopyrite crystal structure. Similar to nanoflowers, larger monodisperse CIS nanospheres with diameters of around 70 nm were formed via the aggregation of many small chalcopyrite CIS NCs. CIS NPLs with different widths (25–35 nm) and a constant length (90 nm) could be formed via tuning the concentration of the metal precursors. The thickness of the CIS NPLs is a few nanometers and the evidence of this, provided by HRTEM images, agrees with the blue-shifted location for their absorption edge due to a weak quantum size confinement effect. In contrast to the nanoflowers and nanospheres which possessed the chalcopyrite structure, the NPLs exhibited a wurtzite crystal structure. The formation mechanism driving the formation of each of these various shapes and morphologies of NCs was investigated through a series of ligand-controlled experiments using different proportions of 1-DDT/t-DDT ligands. The extent of the t-DDT ligand coverage on the initially formed CIS NCs can induce the subsequent aggregation of them to form either nanoflowers of nanospheres at high temperatures. This work provides synthetic pathways for CIS nanostructures with size- and shaped-controlled morphologies, and sheds light on the related formation mechanisms. In the future, the size -controlled synthesis of CIS NPLs and the related optical property studies will be conducted on this interesting heavy-metal free 2D nanostructure.In conclusion, CIS nanostructures with flower-, platelet- and sphere-like shapes were synthesized via a colloidal method, by altering the injected amount of


It has been suggested that speech production is accomplished by an internal forward model, reducing processing activity directed to self-produced speech in the auditory cortex. The current study uses an established N1-suppression paradigm comparing self- and externally initiated natural speech sounds to answer two questions: (1) Are forward predictions generated to process complex speech sounds, such as vowels, initiated via a button press? (2) Are prediction errors regarding self-initiated deviant vowels reflected in the corresponding ERP components? Results confirm an N1-suppression in response to self-initiated speech sounds. Furthermore, our results suggest that predictions leading to the N1-suppression effect are specific, as self-initiated deviant vowels do not elicit an N1-suppression effect. Rather, self-initiated deviant vowels elicit an enhanced N2b and P3a compared to externally generated deviants, externally generated standard, or self-initiated standards, again confirming prediction specificity. Results show that prediction errors are salient in self-initiated auditory speech sounds, which may lead to more efficient error correction in speech production. Speaking is a highly complex human capacity: It does not only involve a motor act, but also leads to the perception and the monitoring of one’s own voice. The distinction between self-produced speech from speech of others is proposed to be accomplished by a “motor-to-sensory discharge”  or an inInterestingly, the suppression effect has been reported in many vocalization studies. It was shown that speech production elicits smaller event-related potentials (ERPs) or fields (ERFs) than passively perceived speech . Based oIn a PET-study, In a further study , we compBased on this study the question arose, if precise predictions are also generated in response to complex auditory stimuli, such as speech sounds. Former studies  which coIn contrast to previous studies, the current study investigated whether predictions are formed on a precise pattern also in response to manually initiated complex, natural sounds , and whether the generation of a precise prediction leads to differential processing of self- compared to externally produced complex deviant stimuli, seen in prediction errors specific to stimuli type. In a first experiment, a standard N1-suppression paradigm was used which has been studied extensively using click sounds, sinusoidal sounds or complex instrumental sounds . Here, wIn a second experiment, we investigated the violation of a precise prediction regarding self-generated complex speech sounds by introducing surprising, unpredictable events, and furthermore we explored the reflection of such prediction errors in the corresponding ERP components. Thus, we modified the N1-suppression paradigm of experiment one corresponding to a former study on self- and externally generated deviant sounds . We compSixteen volunteers  participated in the current study. All participants were right-handed according to the Edinburgh Handedness Inventory . The meaTM TerraTec Edition. Thus, throughout the experiment each participant listened to the vowels that they had previously produced. We asked the participants to produce “ah” and “oh” as in the German words/a:ba/ and/o:ba/. We recorded 10 trials for each vowel per participant. For each participant we picked the vowel that best matched the average characteristics within the 10 trials. Using Praat 5.2.03  we applied minimal normalization procedures to duration, intensity and pitch to maintain natural sound quality. The vowel duration of/a:/was approximately 360.69 ms (SD: 65.66 ms) and pitch around 77.44 Hz (SD: 3.80 Hz). The vowel/o:/had an average duration of 366.31 ms (SD: 77.75 ms) and an average pitch of 80.76 Hz (SD: 3.84 Hz). For both vowels, the sound intensity was calibrated at about 80 dB SPL and an average loudness of 70 dB was maintained by all subjects.In order to obtain individual vowels, we recorded vowel samples from all participants using the program AlgoRecThe first experiment contained two experimental conditions and one control condition  or too fast . The feedback ensured that participants had learned to estimate the time between two successive finger taps without counting. Trials outside the range of 1.8–2.4 s were treated as errors. During the experimental run, no feedback was given.Participants were comfortably seated in an electrically shielded and sound-attenuated experimental chamber. A fixation cross was displayed in the middle of a computer screen. To ensure that the motor activity was comparable across participants in the auditory-motor and the motor-only condition, they were instructed to change hands (index finger) whenever indicated on the screen. Hence all participants tapped in equal parts with left and right hand. The order of tapping hands was randomized across participants. Each tap triggered the instantaneous presentation of a vowel via headphones (Sennheiser HD 202) to both ears in VMC-1/2 and VOC-1/2. An in-house built, highly sensitive tapping device was used to record the finger taps. Participants wore headphones in order to cover them up from all sounds possibly emitted by the taps. In the second experiment, the participants performed a combination of VMC-2 and VOC-2: during one run the standard vowel that was triggered via a tap was the vowel/a:/and the deviant was the vowel/o:/. In the other run, the allocation of standard and deviant vowel was a reversed. The first experiment consisted of 100 trials in each condition. Additionally, we collected 100 trials in MOC-1/2. In the second experiment, we recorded 200 trials in each the VMC-2 and VOC-2 with 70%/a:/as standard and 30%/o:/as deviant and vice versa. In total, 700 trials were recorded in both experiments. Experimental conditions were presented in blocks of 100 trials each. Block order was restricted: The VMC-1/2 always preceded VOC-1/2, but the MOC-1/2 was randomized across participants.The electroencephalogram (EEG) was recorded continuously from 59 Ag–AgCl electrodes according to the International 10–20 system. In addition, activity from the left and right mastoids and the sternum (ground electrode) was recorded. The EEG was sampled at a rate of 500 Hz  and an anti-aliasing filter of 135 Hz was applied. To control for eye movements, vertical and horizontal electrooculograms (EOG) were recorded bipolarly. The impedance of all electrodes was kept below 5 kΩ. The recordings were online referenced to the left mastoid. EEP 3.2.1 Max-Planck-Institute of Cognitive Neuroscience, Leipzig, Germany was used to process the data.Tapping intervals shorter than 1.8 s or longer than 3.0 s were treated as errors, and were excluded from further EEG analysis. We acquired tapping intervals for VMC-1/2 and MOC-1/2 using the Presentation software . For each participant we generated the mean length of the tapped interval per condition and the overall performance accuracy  separately for VMC-1/2 and MOC-1/2.The EEG data were filtered with a 0.3–15 Hz bandpass filter (1601 Hamming windowed filter). The EEG data were re-referenced to linked mastoids. ERPs were time-locked to the stimulus onset of all critical trials. Each analyzed epoch lasted 600 ms including a 100 ms pre-stimulus baseline. The critical epochs were automatically scanned to reject horizontal and vertical eye-movements, muscle artifacts, and electrode drifts. Trials exceeding 30 μV at the eye channels and 40 μV at CZ were rejected. This automatic rejection was corrected manually by applying an eye-movement correction.We controlled for motor activity by computing a difference-wave between VMC-1/2 and MOC-1/2 to compare the sensory activity elicited in the two experimental conditions. This corrected condition was labeled vowel-corrected condition . In the first experiment, we only compared fully predictable self-initiated and externally produced vowels . In the second experiment, we investigated two types of self-initiated vowels – standard (VCS-2) and deviant (VCD-2) vowels, as well as two types of externally produced vowels – self-initiated standard and deviant vowels . As the statistical analysis of the ERP-results in response to standard vowel/a:/and standard vowel/o:/as well as to deviant vowel/a:/and deviant vowel/o:/did not differ significantly, we combined the standard vowels/a:/and/o:/as well as deviant vowel/a:/and/o:/for all further statistical analyses.Group-average ERPs were generated for standards and deviants. In the first experiment, the difference waves  revealed two ERP responses, a negative one in the time window of the N1 peaking at approximately 90 ms, and a positive response in the P2 time window peaking at approx. 190 ms. Statistical analyses were calculated based on individual amplitudes, in the time windows of 70–110 ms for the N1 and 170–210 ms for the P2. In experiment 2, difference waves revealed two ERP responses for standard vowels: a negative one in the N1 time window, peaking at approx. 90 ms followed by a positive one in the P2 time window peaking at approx. 190 ms; and four ERP responses to deviant vowels: a N1 and P2, as well as an N2b peaking at approx. 170 ms and a later positive response in the time window of the P3a peaking at approx. 310 ms. Statistical analyses were calculated based on individual amplitudes in the following time windows: 70–110 ms for the N1, 150–190 ms for the N2b, 170–210 ms for the P2, and 260–360 ms for the P3a. Furthermore, we applied a regions of interest (ROI) analysis using five ROIs .post hoc tests.For the statistical analysis, the SAS 8.20.20  software package was used. Only significant results are presented and where required, the Greenhouse–Geisser correction was applied. Furthermore, we conducted Bonferroni corrected In the first experiment, comparing self-initiated/a:/-vowels to externally produced/a:/-vowels, we ran two analyses of variance (ANOVA), one for each ERP component, including the within-subject factors CONDITION  and ROI . For the statistical analyses of each individual ERP component in the second experiment, we ran a 2 × 2 × 5 ANOVA using the within-subject factors CONDITION , TYPE (standard vs. deviant), and ROI .p = 0.18). In the second experiment, the results were similar. The average tapping interval was 2233.15 ms (SD: 257.43 ms) in VMC-2. Furthermore, participants tapped with an overall correctness of 92.92% (SD: 6.06%). The Kolmogorov–Smirnov-Test revealed a normal distribution (p = 0.82). In MOC-1/2 we found similar results, the average length of the tapping interval was 2388.94 ms (SD: 267.44 ms). Participants performed with an overall accuracy of 93.25% .In the first experiment, the average tapping interval duration was 2469.79 ms (SD: 360.77 ms) in VMC-1. Furthermore, participants tapped with an overall accuracy of 85.80% (SD: 18.73%). The Kolmogorov–Smirnov-Test revealed no deviance from the normal distribution  .Experiment 1 revealed the expected N1-suppression effect as well as a reduced P2 in response to self-initiated vowels.All statistical results are presented in In the N1 time window, the ANOVA revealed significant differences between condition and region. According to our hypothesis, we conducted a planned analysis, investigating condition effects within the different vowel types: In the standard vowels  we foundThe results show that we only find a N1-suppression effect in response to self-initiated standard.In the P2 time window, the ANOVA analysis revealed a significant difference between conditions – self-initiated versus externally initiated – and types – standard versus deviants, as well as significant interactions between condition and region aa well as a marginally significant effect between condition and type. In response to standard vowels , we founTaken together, the results show a significant P2-reduction in response to self-initiated standard sounds compared to externally produced standards. Although externally produced deviant and standard vowels elicited a similar P2 effect, a possible P2 in response to self-initiated deviant vowels cannot be statistically evaluated due to potential overlay effects.post hoc analysis of condition, revealed a significant difference between vowel types, as well as a significant interaction between vowel type and region, showing that only self-initiated deviant vowels elicited an N2b effect .In the N2b time window, we found a significant difference between conditions  and types , as well as a significant interaction between condition and region, type and region, condition and type, and condition, type and region. The In contrast , we did In conclusion, the N2b was only elicited in response to self-initiated deviant vowels.F = 22.68; p = 0.0005]. In a post hoc analysis we resolved the type effect, analyzing standard and deviant sounds separately. We found a significant difference between the two conditions in the deviant vowels , revealing a significantly enhanced P3a in response to self-initiated deviant vowels (VCC-2 mean amplitude: 1.60 μV) compared to externally produced deviant vowels (VOC-2 mean amplitude:0.64 μV)  . However0.64 μV) . The resThe current study investigated the questions whether precise predictions are generated to process self-initiated complex speech sounds  by applying an internal forward model and whether prediction errors regarding self-generated deviant vowels are reflected in corresponding ERP components. In order to address these questions, two experiments were conducted. In the first experiment, we used a standard N1-suppression paradigm comparing self- and externally produced individually pre-recorded vowels in order to test whether the N1-suppression paradigm suffices to investigate complex auditory stimuli such as speech sounds. The results revealed a strong N1-suppression effect in response to self-initiated vowels. This finding confirms the successful generation of a forward prediction independent of the complexity of an anticipated stimulus.Moreover, we found a reduced P2 response elicited by self-initiated vowels. Although the literature is very diverse regarding the P2, with some studies showing a suppression effect , whereasInterestingly, a recent study  testing In the second experiment, we adapted the N1-suppression paradigm by comparing self- and externally produced standard and deviant vowels. The deviant vowels (30%) were either an/a:/or an/o:/dependent on which of these two vowels represented the standard vowel. Comparable to the results of the first experiment, standard vowels elicited a suppressed N1 and P2 component in response the self-initiated vowels indicating that motor-to-auditory predictions are generated in order to process self-initiated vowels. Self-initiated deviant vowels, on the other hand, did not elicit a clear N1-suppression effect, indicating the violation of a precise prediction. In contrast, externally produced deviants did not differ from externally produced standard vowels showing that the difference in the N1-suppression effect was not elicited due to deviant detection. Additionally, our results show that the P2 in response to deviant vowels reveals a more complicated pattern: The P2 elicited by externally produced deviants is well pronounced and very similar to the P2 in response to externally produced standard vowels. In contrast, the potential P2 component in response to self-initiated deviant vowels is overlaid by an N2b response , and canFurthermore, deviant vowels elicited an N2b effect, which suggests the conscious detection of an unexpected, infrequent stimulus . The effThe results of the current study replicated the pattern of components found in a previous study on deviancy processing in sinusoidal sounds . This stHowever as we hypothesized that the N1-suppression effect reflects the precision of the prediction, we modulated the standard paradigm introducing deviant vowels (second experiment) to reduce the prior knowledge concerning an anticipated stimulus and engenders a prediction error. The results show that precise predictions are generated  as the NThe assumption that precise predictions are generated also in response to complex self-initiated stimuli receives further support from the enhanced N2b effect in response to self-initiated deviant vowels. Similar to our previous study , we consThe results concerning the detection of deviants nicely complements the results presented in the literature on speech monitoring and processing of speech errors  which moIn the current study, we have investigated motor-to-auditory predictions, framed within a forward model account, which is specific to motor control and uses the concept of a efference copy to generate a prediction. Other current accounts of surprising  responses in a more general, modality-independent framework are usually cast in terms of predictive coding e.g., . In thesIn conclusion, the present study presents two experiments: The first investigates whether forward predictions are generated to process self-initiated complex speech sounds ? And the second addresses the question whether prediction error regarding self-initiated deviant, are these predictions based on precise patterns, as in holding a concrete representation of the anticipated vowel? Addressing the first question, the results revealed N1 and P2 suppression elicited by self-initiated vowels, indicating a successful generation of predictions in response to complex self-initiated speech sounds. This finding supports the notion that processing of self-produced speech mirrors components of a forward model, preparing respective cortical areas for incoming sensory consequences of self-produced speech. Investigating the second question, we found N1-suppression in response to self-initiated vowels compared to externally generated vowels. In addition, we report an enhanced N2b and P3 effect in response to self-initiated compared to externally produced deviant vowels. These findings imply that specific predictions are generated. Furthermore, we show that prediction errors are more salient in self-initiated speech sounds compared to externally produced sounds. More generally, our results speak to a key role of agency in predictive processing formulations; namely, a key role in mediating sensory attention and its reversal when attending to the consequences of self-generated acts.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the Leipzig University, Leipzig, Germany. The patients/participants provided their written informed consent to participate in this study.FK, SK, and ES developed the design and concept. FK collected and analyzed the data. MS contributed to the analysis. FK wrote the first draft. FK, SK, ES, and MS discussed the results and edited draft for final submission. All authors have approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli was intranasally instilled to reproduce non-fatal acute pneumonia model. After infection, serum samples and lung tissues were obtained at 0, 12, 24, and 72 h. DIO mice exhibited increased serum triglyceride (TG) and total cholesterol (TC) contents as well as pulmonary resistin, IL-6, and leptin levels compared with lean mice. E. coli infection caused an acute suppurative inflammation in the lung with increased lung index and serum TG and TC contents; elevated pulmonary tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, and leptin levels; and oxidative stress in mice. Interestingly, almost all the above-mentioned parameters peaked at 12 h after infection in the lean-E. coli group but after 12 h in the DIO-E. coli group. These results indicated that the DIO mice presented a delayed inflammatory response and oxidative stress in non-fatal acute pneumonia induced by E. coli infection.Obesity has been identified as a risk factor for adverse outcomes of various diseases. However, information regarding the difference between the response of obese and normal subjects to pulmonary inflammation is limited. Mice were fed with the control or high-fat diet to establish the lean and diet-induced obese (DIO) mice.  Mucins (glycosylated proteins) produced by epithelial tissues in the air conduction part of the lungs play an important role in defense against foreign bodies and pathogenic microorganisms and are overexpressed in several lung diseases2.The lungs are the primary organ of the respiratory system in humans and many other animals. They are responsible for gas exchange in the respiratory system. The elasticity and tensile strength of the lungs are ensured by their connective tissue networkEscherichia coli, a gram-negative bacillus, is a kind of respiratory pathogen and strong inducer of pro-inflammatory cytokine, and it can cause nosocomial pneumonia3. Moreover, sublethal intratracheal challenges of E. coli in mice result in the migration of neutrophils into the pulmonary air space4. Neutrophils that infiltrate the lungs and migrate into the airways express pro-inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and appear to contribute to oxidant-induced injury and loss of epithelial integrity5.6. Obesity increases morbidity and mortality from many chronic health ailments, such as cardiovascular disease, type II diabetes, dyslipidemia, and fatty-liver disease7. Obesity also increases the risk of respiratory tract infections, such as pneumonia8. However, paradoxically, recent studies reported that obese patients show improved outcome such as reduced mortality in acute bacterial pneumonia11. In obesity, the ability of the adipose tissue to elaborate cytokines and adipocytokines, such as IL-6, TNF-α, leptin, and resistin, is increased to possess pro-inflammatory properties. The low-grade chronic systemic inflammation associated with obesity can potentially enhance pulmonary immune responses against respiratory infections12.Obesity is a medical condition in which excess body fat has accumulated to the extent that may pose a negative effect on health. Worldwide obesity has nearly tripled since 197513. It will be enthralling to shed light on how the lung is affected by acute bacterial pneumonia in obese models from earlier reports of pneumonia. To understand this concept, we selected ICR mice fed with high-fat diet to establish the obese model. We instilled E. coli intranasally in mice to establish an acute nonfatal bacterial pneumonia model. Subsequently, changes in histopathology and cytokines, adipocytokine secretion, and oxidative stress status were detected to investigate the impact of obesity on nonfatal pneumonia induced by E. coli.The reasons of these findings are unclear. One potential factor is the alteration of pulmonary immune responses in the obese, which may lead to a heightened state of host defensep < 0.05) than that of the lean mice. During the experiment, the body weight of the DIO groups were higher than that of the lean groups (p < 0.05). After infection, the body weight in the lean-E. coli group and the DIO-E. coli group showed a decreasing tendency compared with the uninfected groups . After being infected with E. coli, the serum TG and TC contents in the DIO-E. coli group significantly increased at 72 h compared with the DIO-uninfected group. In the lean-E. coli group, only the serum TC content markedly increased at 12 h in comparison to the lean-uninfected group (p < 0.05).As shown in Fig. E. coli, the lung weight was higher in the DIO group than in the lean group (p < 0.05). After being infected with E. coli, the lung weight significantly increased in the lean-E. coli group compared with the lean-uninfected group from 12 h to 72 h. Interestingly, we only detected a significant increase (p < 0.05) of lung weight in the DIO-E. coli group at 72 h in comparison to the DIO-uninfected group. Moreover, the lung index showed no significant difference at 0 h (p > 0.05). During the whole 72 h post-infection, increased lung index was detected in the E. coli-infected groups compared with the uninfected groups (p < 0.05)  Fig. .Figure 3E. coli, the texture of the affected areas in the superior lobes of the left and right lungs became hard with a dark red or grey appearance. The affected area in the DIO-E. coli group was smaller than that in the lean-E. coli group.Figure E. coli infection, typical acute suppurative inflammation was noted in the lean- and DIO-E. coli groups, as shown in Fig. E. coli group were lesser than that in the lean-E. coli group. However, serious congestion and hemorrhage in alveolus occurred in the DIO-E. coli group compared with the lean-E. coli group, which even caused the hemosiderin granules to deposit in the hemorrhage areas. At 24–72 h after infection, the severity of neutrophil infiltrate condition and incidence of congestion and hemorrhage were decreased in the lean- and DIO-E. coli group.Before infection, the normal histology of conducting airway structure (bronchi and bronchioles) and gas exchange area  was microscopically observed in each group. As shown in Table E. coli, the occurrence rate of acid mucins increased sharply in the lean-E. coli group compared with the DIO-E. coli group. No neutral mucin was detected in the uninfected groups, whereas the occurrence rate of neutral mucins was increased in the E. coli-infected groups. Moreover, the occurrence rate of neutral mucin peaked at 24 h in the lean-E. coli group but showed a continuous increasing trend in the DIO-E. coli group from 12 h to 72 h.The mucin occurred in the goblet cell and mucous gland of the respiratory tract. As shown in Fig. p < 0.05). After infection, the integrated optical density (IOD) of collagen fibers showed a decreased tendency in the lean-E. coli group and DIO-E. coli group in comparison to each uninfected group. However, the significant decrease was detected only at 24 and 72 h in the DIO-E. coli group (p < 0.05).The collagen fibers were mainly located in the loose connective tissue below the epithelium and oriented circularly or obliquely around the bronchiole. As shown in Fig. E. coli, the DIO groups had significantly higher contents of IL-6, leptin, and resistin but lower content of IL-8 compared with the lean groups (p < 0.05).The contents of pulmonary cytokines and adipocytokines are shown in Fig. E. coli, the content of pulmonary TNF-α was significantly higher (p < 0.05) in the lean-E. coli group compared with the lean-uninfected group. However, in the DIO-E. coli group, the content was significantly increased (p < 0.05) only at 24 h compared with the DIO-uninfected group  at 12 and 24 h compared with each uninfected group. Moreover, this content was significantly higher in the lean-E. coli group than in the DIO-E. coli group (p < 0.05) at 12 h . By contrast, the pulmonary IL-6 content was significantly increased (p < 0.05) only at 12 h in the DIO-E. coli group compared with the DIO-uninfected group  in the E. coli-infected groups  in the E. coli-infected groups compared with the uninfected groups  at 12 to 72 h in the E. coli-infected groups compared with the uninfected groups. The resistin contents in the DIO-E. coli group dramatically decreased (p < 0.05) compared with the DIO-uninfected group at all time points .To evaluate the state of oxidative stress, the contents of glutathione (GSH) and malonaldehyde (MDA) and the activities of GSH peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) were measured Fig. . Before p < 0.05) at 12 and 72 h in the lean-E. coli group compared with the lean-uninfected group. However, in the DIO-E. coli group, the GSH content increased only at 12 h (p < 0.05) and then decreased from 24 h to 72 h compared with the DIO-uninfected group   Fig. .p < 0.05) in the E. coli-infected groups at 12 h after infection and then significantly declined (p < 0.05) from 24 h to 72 h in the lean-E. coli group and at 72 h in the DIO-E. coli group . The SOD activity significantly increased (p < 0.05) in the DIO-E. coli group at 12 and 24 h and then showed a decreasing tendency. However, no significance was found (p > 0.05)  in the E. coli-infected groups from 12 h to 72 h compared with the uninfected groups  Fig. . The actups Fig. .E. coli. Moreover, in the lean mice, after these parameters markedly increased at 12 h, they declined at 24 h and started to level off from 24 h to 72 h. However, the fluctuation of these parameters in the DIO mice was more irregular.In conclusion, the most changeable factors in the lean and DIO mice were GSH and GSH-Px. The lean mice showed more dramatic change than the DIO mice after being infected with 14. In accordance with previous studies16, after feeding with a high-fat diet for 8 weeks, the serum TG and TC levels in the DIO mice were significantly increased compared with those in the lean mice, indicating a higher metabolic reserve in DIO mice. Moreover, in our previous study17, a different dosage of E. coli was intranasally instilled to establish bacterial pneumonia. Unpredictably, we found that during mild infections , obesity improved host defenses against infection, promoting recovery. However, in severe infection , obesity exerted negative effects on host defenses. To understand the protective effect of obesity on lung injury and inflammatory response of mice affecting milder infections, 109 CFUs/mL of E. coli was used in the present study.Obesity is essentially caused by an imbalance in energy intake and expenditure18. E. coli possesses abundant lipopolysaccharide (LPS). The increase in serum TG and TC levels induced by LPS administration may be related to the stimulation of hepatic lipogenesis and adipose tissue lipolysis, lipoprotein lipase activity, decreased TG clearance19, and increased low-density lipoprotein cholesterol20. Our results also showed that after infection with E. coli, the levels of serum TG and TC increased at 12 h in the lean-E. coli group, whereas its increase was delayed to 72 h in the DIO-E. coli group. These phenomena were similar to pneumonia patients with normal weight who may not have enough metabolic reserve to counteract the increased catabolic stress21. Cytokines, especially TNF-α and IL-1β, can acutely increase serum cholesterol and TG metabolism22. Interestingly, in the present study, the levels of pulmonary TNF-α and IL-1β, particularly the former, increased sharply in the lean-E. coli group compared with the DIO-E. coli group at 12 h after infection. Therefore, the dramatic changes in the TG and TC contents in the lean-E. coli group may have been, at least partially, attributed to the higher levels of TNF-α and IL-1β than those in the DIO-E. coli group.Metabolic alteration occurs during infection/inflammation, such as increased serum TG and TC levels in rodentsE. coli is one of the main causes for bacterial pneumonia episodes worldwide23. Once E. coli invades the alveoli, it triggers the immune system to respond by sending white blood cells responsible for attacking microorganisms to the lungs and then releasing cytokines to further activate the immune system24. In the present study, after infection with E. coli, both the DIO- and lean-E. coli groups infiltrated large numbers of inflammatory cells, which led to a significant increase in lung weight and lung index in the E. coli-infected groups. Neutrophils are characteristic of acute bacterial suppurative pneumonia, expressing pro-inflammatory cytokines26. IL-8 is characterized as a neutrophil chemotactic factor in a variety of lung diseases and can cause the recruitment of neutrophils to the pulmonary interstitium and airspace27. Alveolar macrophage can destroy bacteria and release various inflammatory cytokines, such as IL-1, IL-6, and TNF-α30. As revealed by pulmonary cytokine detection, the increase in pulmonary TNF-α, IL-1β, IL-6, and IL-8 levels was more severe in the lean mice than in the DIO mice after infection. This finding was consistent with the severity of inflammatory cell infiltration in histopathological observation. Moreover, as reported previously, mice fed a high-fat diet showed increased vascular permeability and vascular dysfunction31, and the chronic low-grade inflammation of obesity impaired pulmonary vascular homeostasis and enhanced susceptibility to acute injury32. Our results demonstrated that the DIO mice exhibited higher incidence of congestion before infection and showed more serious hemorrhage after E. coli infection than the lean mice, indicating an increased vascular permeability in DIO mice. Therefore, even though the DIO mice were less sensitive to recruit inflammatory cells, they showed more serious damage in pulmonary vasculature than the lean mice.1. The long-term chronic inflammation status in obesity indicates excessive fibrosis deposited in the connective tissue33. Pulmonary fibrosis is also associated with increased expression of collagens in connective tissue34. Similar to a previous report33, our results revealed that DIO mice with high collagen fiber density may increase the risk of pulmonary fibrosis. Moreover, the adventitial connective tissue of bronchioles is primarily loose, with many collagen and elastic fibers oriented longitudinally or perpendicularly to the long axis of the airway35, and the wavy structure of collagen fiber provides the lung with a substantial degree of extensibility1. In the present study, after infection, inflammatory cells and mucous exudate infiltrated into the adventitial connective tissue of bronchioles and destroyed the wavy structure of collagen fibers in the lung. In addition, by modulating T-cell response, leptin may play a protective role in accumulating pulmonary collagen during inflammation37. In the present study, the mice in the E. coli-infected groups that possessed high levels of leptin exhibited decreased density of pulmonary collagen fibers. Mucins are a family of heavily glycosylated proteins (glycoconjugates) produced by epithelial tissues in most animals, and their key characteristic is to form chemical barriers and bind to pathogens as a part of the immune system2. Previous studies detected overexpressed mucins in various lung diseases, such as asthma, bronchitis, chronic obstructive pulmonary disease, and cystic fibrosis39. Similar to these studies, the secretion of mucins in epithelial cells of the intrapulmonary conducting airways was also increased in the infected groups. Interestingly, the increased secretion of mucins in the lung was greater in the lean-E. coli group at 12 and 24 h; however, in the DIO-E. coli group it was observed at 72 h.Elastin and collagen are the major components of the lung connective tissue network, providing the lung with elasticity and tensile strength41 and shows complex effects on immune function and inflammation with pro- and anti-inflammatory elements42. As previously reported, leptin-deficient mice (ob/ob mice) exhibit reduced lung neutrophil counts and levels of pro-inflammatory cytokines during pneumonia44, and obese patients with pneumonia show low pneumonia severity index scores and plasma levels of C-reactive protein45. Similar to these reports, in our study, the fold change of cytokine contents, especially TNF-α and IL-6, in the lean-E. coli group was higher than that in the DIO-E. coli group, which might be attributed to increased pulmonary neutrophil and macrophage infiltration in the lean-E. coli group after infection. Moreover, obese individuals with high serum cholesterol concentrations can neutralize circulating LPS, therefore decreasing inflammation46. Our results revealed high serum TG and TC levels in the DIO mice, which might exhibit lower fold change of adipocytokine and cytokine contents after infection. In addition, resistin is a pro-inflammatory protein and serves as a marker of disease severity and a mediator of prolonged inflammatory state47. In the present study, the pulmonary resistin level was down-regulated in the DIO mice, which was one marker of mild infection status, as well as a delayed inflammatory response.Obesity is associated with a chronic inflammatory response attributed to the secretion of adipocytokines and various other cytokines49. GSH-Px is a peroxidase protecting the organism from oxidative damage50. Obesity is associated with an increase in oxidative stress and formation of reactive oxygen species (ROS)51. Adipocytokines can induce the production of ROS, generating a process known as oxidative stress52. Based on our results, the DIO mice showed higher MDA contents, GSH-Px activity, and adipocytokine  levels than the lean mice, indicating that DIO mice suffered from high oxidative stress levels before infection.Oxidative stress plays critical roles in the pathogenesis of various diseases. MDA is a marker for oxidative stress and the end product of lipid oxidation53. In our present study, the content of oxidative product (MDA) and antioxidants (GSH) and activity of antioxidase  were increased in the infected groups at 12 h after infection. Moreover, GSH is an important antioxidant that is capable of preventing damage from ROS under the catalysis of GSH-Px54. In this study, the two most increased parameters in the infected group were GSH content and GSH-Px activity, which attempted to prevent the lung from oxidative damage. In addition, obesity can induce the down-regulation of antioxidant enzyme activities55. To combat infection, different types of cells  rapidly release large numbers of ROS and lysosomes to destroy bacteria56. In the present research, the fold change of antioxidant parameter contents was lower in the DIO-E. coli group than in the lean-E. coli group, which was attributed to the status of obesity and low numbers of infiltrated neutrophils in DIO mice. These antioxidant parameters started to level off from 24 h to 72 h in the lean-E. coli group but were still irregular in the DIO-E. coli group. Therefore, when the excess lipid oxidation induced by infection continued, obesity-induced oxidoreductase diminution would ultimately cause the increased generation of the lipid peroxide, such as MDA, at 72 h in the DIO-E. coli group.The phagocytosis of gram-negative bacteria can activate the primary host defense mechanism and result in the generation and release of ROSE. coli-infection induced an acute suppurative inflammation in the lungs and increased lung weight and lung index, cytokine and adipocytokine levels, and oxidative stress levels in the E. coli-infected groups. Interestingly, the DIO mice with high levels of metabolic reserve and adipocytokines exhibited a delayed inflammatory response. Metabolic disorder, neutrophil infiltration, inflammatory factor generation, and increased MDA levels in the lean-E. coli group were more important than those in the DIO-E. coli group at 12 h; however, these effects were reversed at 72 h. These results indicated that the DIO mice presented a delayed inflammatory response and oxidative stress to the non-fatal acute pneumonia induced by E. coli infection.In conclusion, ad libitum. Based on a previous study17, after feeding with high-fat diets for 8 weeks, mice were weighed, and their height was measured. Mice with obese index exceeding 20% and significantly high Lee’s index were considered as obese animals.A total of 256 male ICR mice (21 days old) were purchased from the Dossy Animal Center  and housed under specific-pathogen-free condition. All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at Sichuan Agricultural University . The mice received either a normal diet or a high-fat diet, which were both purchased from the Dossy Animal Center. Food and water were supplied All experimental procedures were performed in accordance with the national and international guidelines and regulations and were approved by Sichuan Agricultural University Animal Care and Use Committee .E. coli obtained from Veterinary Medical Laboratory of Sichuan Agricultural University  was cultured in Luria-Bertani broth  at 37 °C for 18 h. Then, the bacterial culture was centrifuged, and bacterial pellets were resuspended in phosphate-buffered saline (PBS) to produce the inoculums.E. coli, namely, lean-uninfected, DIO-uninfected, lean-E. coli, and DIO-E. coli groups.After 8 weeks, the mice fed with either normal or high-fat diets were divided into two groups (128/group) as lean and DIO groups, respectively. Then, the two groups were subdivided into four groups (64/group) after being intranasally instilled with PBS or E. coli and DIO-E. coli groups were quickly anesthetized with ether and intranasally instilled with 40 μL of E. coli inoculum  suspended in PBS according to a previous study17. The same amount of PBS was given to the mice in the lean-uninfected group and DIO-uninfected group by the same method. Animals were monitored closely for 72 h after E. coli infection. After intranasal infection with E. coli or PBS, mice were sacrificed under anesthesia at 0 (pre-infection), 12, 24, and 72 h (post-infection), and the lungs were harvested and prepared for the following analyses.The mice in the lean-E. coli infection. After necropsy, the lungs were collected without connective tissues and weighed. The lung index was calculated using the following formula:During the experiment, no mouse died after Blood samples were obtained retro-orbitally from eight mice in each group, and individual sera were separated via centrifugation and stored at −20 °C. TG and TC concentrations in the serum were determined using a commercially available kit .Eight mice from each group were euthanized. The lungs were observed and photographed and then immediately fixed in 4% paraformaldehyde. Subsequently, they were dehydrated in alcohol, embedded with paraffin, sectioned at 5 μm, and processed for hematoxylin and eosin staining and the following specific histological stainings: Masson’s trichrome for fiber and AB-PAS for mucins. Histopathological changes were observed and photographed with a digital camera . By Masson’s trichrome staining, collagen fiber stains blue, erythrocyte stains red, and cell nucleus stains black. By AB-PAS staining, neutral mucins stain red and acid mucins stain blue. The same position of the lungs in eight mice was observed through a microscope, and the histological lesion of the lung was evaluated through the incidence of congestion and hemorrhage, severity scoring of neutrophil infiltrate, and proportion of airspace areas. The level of severity was judged from − to ++++, which represented none to severe. The airspace proportion was the ratio of the airspace area and the total area of one view under 400× magnification.The total number of bronchioles in eight mice was counted through a microscope, and the number of bronchioles that contained neutral or acid mucins was recorded. The occurrence rate of mucins  was the ratio of the mucin-stained bronchiole number and the total bronchiole number, which was calculated to reflect the mucin content in the lung. The Integrated Optical Density (IOD) of collagen fibers was measured to represent the collagen fiber contents of lung. The airspace area proportion and IOD of collagen fibers were analyzed using image-Pro Plus 6.0 (USA).At the indicated time points, eight mice in each group were euthanized, and the lungs were immediately collected for evaluating the contents of several cytokines and adipocytokines. The lung (1 g) was homogenized with normal saline buffer (9 mL) through a cell homogenizer in an ice bath and centrifuged at 3,000 r/min for 10 min to obtain a clear supernatant. TNF-α, IL-1β, IL-6, IL-8, leptin, and resistin concentrations in pulmonary supernatants of mice in each group were measured with mouse ELISA kits  according to the manufacturer’s instructions.2O2 decomposition rate. The values were expressed as nmol/mg protein for GSH and MDA and units (U)/mg or U/g of protein for GSH-Px, SOD, and CAT.After determining the concentration of total protein in the supernatant of the pulmonary homogenate by the bicinchoninic acid method, the GSH and MDA contents and GSH-Px, (SOD), and CAT activities were measured by biochemical method following the instruction of reagent kits . The GSH assay was based on the development of a yellow color when 5,5′-dithio-bis-nitrobenzoic acid was added to compounds containing sulfhydryl groups. The MDA assay was determined by the thiobarbituric acid colorimetric method. The GSH-Px activity was detected by the consumption of GSH. The SOD activity was determined by the xanthine oxidase method. The CAT activity was determined by the Ht test, whereas the significant differences among four groups within 72 h experiment were analyzed by variance analyses (LSD or Dunnett’s T3). The results were expressed as means ± standard deviation. The analyses were performed using SPSS 17.0 software  for Windows. Statistical significance was considered at p < 0.05.The significance of difference between two groups was analyzed by the independent samples


In this paper, water-soluble 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose with a high degree of oxidation was prepared by a two-step process using bamboo dissolving pulp. The first step was to destroy the cellulose crystal I by NaOH/urea solution to obtain cellulose powder with decreased crystallinity. The second step was to oxidize the cellulose powder by TEMPO oxidation. The TEMPO-oxidized cellulose was analyzed by Fourier transform infrared spectroscopy (FTIR), conductimetry, X-ray diffraction (XRD), fiber analyzer, and transmission electron microscopy (TEM). FTIR showed that the hydroxymethyl groups in cellulose chains were converted into carboxyl groups. The degree of oxidation measured by conductimetry titration was as high as 91.0%. The TEMPO-oxidized cellulose was soluble in water for valuable polyelectrolytes and intermediates. Cellulose is one of the most luxuriant natural macromolecules in the world. As one of the high crystalline structural polysaccharides, cellulose has attracted extensive attention due to its high availability, biodegradability, biocompatibility, and good mechanical properties ,2. Cellud-glucan chains. However, the hydroxyl groups on one chain form hydrogen bonds with nearby oxygen to construct stable and rigid molecules, making it insoluble in common solvents, which hinder certain applications of cellulose. Various modifications have been developed to improve the solubility and expand the application of cellulose [d-glucose unit into charged carboxyl entities region-selectively. That is, only the hydroxymethyl groups of polysaccharides are oxidized, while the secondary hydroxyls remain unaffected. Individual cellulose nanofibers, 3–4 nm wide and a few microns long, were prepared through a combined TEMPO oxidation and mechanical treatment [Each cellulose is composed of multiple linear -linked β-ellulose ,11,12,13ellulose ,26,27,28reatment .The success of using TEMPO/NaBr/NaClO oxidation to produce water-soluble polyglucuronic acid strongly depends on the accessibility and on the crystalline state of the starting material ,18. WhenBamboo dissolving pulp is a promising alternative non-wood material owing to the high growth rate of bamboo, low resource cost, long or semi-long fibers, and similar α-cellulose content compared to most trees . HoweverIn this paper, a water-soluble TEMPO-oxidized cellulose with a high degree of oxidation was prepared using bamboo dissolving pulp by a two-step process. During the first step, the cellulose powder with decreased crystallinity was obtained by destroying the cellulose I structure using a NaOH/urea solution. In the second step, cellulose powder was oxidized by TEMPO/NaBr/NaClO to obtain carboxylated cellulose with a high degree of oxidation and high carboxyl content.The cellulose was obtained from a bamboo dissolving pulp board, purchased from Sichuan Tianzhu Resources Development Co., Ltd., Sichuan, China. The α-cellulose content of the pulp was 94.5 wt %, the hemicellulose content was 5.2 wt % and the ash content was 0.1 wt %. The weight average molecular weight measured by gel permeation chromatography was 141,000 and the number average molecular weight was 56,000. TEMPO, sodium hypochlorite (NaClO), sodium bromide (NaBr) sodium hydroxide (NaOH), urea, and ethanol were supplied by Sinopharm Group Chemical Reagent Co., Ltd., Tianjin, China. All reagents were analytical grade and used without further purification.Ten grams of bamboo dissolving pulp were dissolved into 2 L of NaOH/urea/water solution (7:12:81 by weight) at −12 °C under vigorous stirring ,31. AfteThen, 4.8 g of NaOH/urea-treated cellulose were dispersed in 500 mL of deionized water, and 0.16 g of TEMPO and 12.70 g of NaBr were added. The pH of suspension was controlled at 10 by 0.1 mol/L NaOH solution (monitored with a pH meter). The TEMPO-mediated oxidation of the cellulose powder was initiated by adding 114 mL of 8 wt % NaClO solution dropwise in 10 min and conducted at room temperature under gentle agitation. The solution was reacted for 6 h and the pH was maintained at 10 ± 0.1. The reaction was terminated by adding 5 mL of anhydrous ethanol. The solution was dialyzed in deionized water for two days. The final product of NaOH/urea-treated TEMPO-oxidized cellulose was obtained after freeze-drying.For comparison, the bamboo dissolving pulp was oxidized directly by TEMPO-mediated oxidization to obtain direct TEMPO-oxidized cellulose . BrieflyC is the NaOH concentration, V1 and V2 are the volume of NaOH, and m is the weight of the oven-dried sample.DO was expressed as the ratio between the amount of oxidized hydroxymethyl groups and total hydroxymethyl groups, which was determined by conductimetric titration. A 50-mg cellulose sample was suspended/dissolved into 15 mL of 0.01 mol/L hydrochloric acid solutions. After 10 min of stirring, the suspension was titrated with 0.005 mol/L NaOH solution under stirring. The conductivity was monitored using a conductivity meter throughout the titration process. The titration was terminated when the pH reached 11. DO was calculated by the following equation ,19:DO=16−1 with a resolution of 4 cm−1.Infrared spectra were recorded on an FTIR  spectrometer. Oxidized samples were converted to their acid form in order to displace the carboxyl absorption band toward higher wavelength. The samples were dried in an oven and analyzed as KBr pellets. All spectra were recorded in the wave number ranging from 4000 to 400 cmThe fiber morphologies of different cellulose samples were investigated by a fiber analyzer . For this analysis, 0.2 g of the sample was dispersed in 1000 mL ethanol.CrI), which could be calculated according to the following equation [I200 is the diffractogram height of (200) peak , and Iam is the height of the amorphous background .The crystalline structures of different cellulose samples were investigated by a wide angle X-ray diffraction  with Cu Kα radiation at the wavelength 1.5405 Å. The continuous scanning angle range used in this study was 5–60°, and the machine was operated at 20 kV and 5 mA. The relative degree of crystallinity was obtained from the crystallinity index . The solution was dropped on the grid and dried in air. The TEM measurements were performed using an FEI Tecnai G2 F20  equipped with a CCD camera  operated at 200 kV.The NaOH/urea-treated TEMPO-oxidized cellulose with a high degree of oxidation was prepared using a two-step process, as shown in −1 is assigned to C–O stretching of the cellulose backbone and is not influenced by TEMPO-mediated oxidation. This peak can be treated as the internal standard [−1, which corresponds to the C=O stretching vibration of carboxyl groups in their acidic form. This suggests that the hydroxymethyl groups of the d-glucose unit were converted into carboxyl groups successfully. The intensity of NaOH/urea-treated TEMPO-oxidized cellulose is stronger than that of direct TEMPO-oxidized cellulose, indicating a higher amount of carboxyl groups and a higher degree of oxidation.FTIR spectra of cellulose before and after oxidation were shown in standard ,34. Fromθ = 14.8°, 16.4°, 22.6°, and 34.2° are indexed as  planes . For the crystal . These rThe crystallinity of cellulose before and after oxidation was calculated according to the X-ray diffraction pattern, as shown in The DO of cellulose was determined from the conductometric titration. The conductivity versus the volume of NaOH aqueous solution was plotted in The NaOH/urea-treated TEMPO-oxidized cellulose dissolves in water easily, but not in ethanol. To study the morphology of cellulose before and after oxidation, all the samples were dispersed in ethanol and observed by a fiber analyzer. The pristine cellulose in To further determine the dimension of NaOH/urea-treated TEMPO-oxidized cellulose, TEM observation was performed. The inset of A two-step process was demonstrated to prepare water-soluble TEMPO-oxidized cellulose with a high degree of oxidation using bamboo dissolving pulp as the starting material. After destroying the hydrogen bonds in cellulose I during the first step, the cellulose powder with decreased crystallinity was oxidized by TEMPO/NaBr/NaClO to obtain carboxylated cellulose with a high degree of oxidation during the second step. The TEMPO-oxidized cellulose is soluble in water for valuable polyelectrolytes and intermediates with many possible functionalities.


The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L−1. To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems.Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu This mass range includes relatively low masses which results in a more complex mass spectrum, due to the presence of matrix related compounds + Adda and the level of identification is set at level 2 since a certified standard would have confirmed the identification of the compound. We have identified this compound as MC-HtyR, and this is to our knowledge the first time this compound was identified MC-LR in sample no. 3 1]le no. 3 , MC-LR  in samplle no. 9 . The thrA second identification workflow to identify APs was tested for the elucidation of AP-A found in sample no. 5 and confirmed with a certified standard, shown in m/z 84.08136, an immonium fragment ion of lysine. Using this fragment ion alone, the features lists were reduced significantly, with 0 to 8 possible candidates identified with exact masses alone , were used to identify AA1 (1-CO-Lys2-Met3-Leu4-MeIle5-Met(O)6 according to fragmentation spectra  according to previously published method MC-RR and MC-RR were predominant in the two European samples (samples no. 11 and 12) with the highest concentrations being 41,000 and 5700 ng L−1 and MC-LA was also predominant in three samples  with concentrations varying between 364 and 1165 ng L−1. In addition, AP-A and AP-B were found in half of the samples with concentrations varying from 95 up to 6000 ng L−1. These two APs were also predominant in two samples (5 and 6), and their ubiquity is supported by previous studies  MC-LR and MC-RR (purity ≥ 99%) were purchased from the National Research Council of Canada . Nodularin-R (NOD-R), [Asp3] MC-RR, MC-YR, LA, LY, LW, LF, WR, HtyR and HilR (purity ≥ 95%) were purchased from Enzo Life Science . Homoanatoxin-a  was obtained from Abraxis, Inc. , Anabaenopeptin A and B  from Cyano Biotech GmbH , and 15N10-MC-LR (95%) from Cambridge Isotopes Laboratories, Inc. . Individual stock solutions of ANA-a, CYN, MC-LR, [Asp3] MC-LR and MC-RR were kept at −20 °C for a maximum of six months. All other individual stock solutions were prepared in methanol (MeOH) at a concentration of 25 mg L−1 and were kept at −20 °C for a maximum of one year. Primary working solutions were prepared at a concentration of 100 µg L−1 for targeted cyanotoxins and 9 µg L−1 for internal standards (Iss: 15N10-MC-LR and NOD-R) by dilution in MeOH of individual stock solution aliquots. Subsequent working solutions were prepared daily by dilution in water to give solutions of the desired concentration. All organic solvents and water used for dilutions were of HPLC grade purity from Fisher Scientific . ANA-a, CYN, MC-LR, [AspTM NalgeneTM, Waltham, MA, USA), previously rinsed three times with the surface water from the site [−1). Prior to quantitative analysis, the internal standards were added for a final concentration of 300 ng L−1. Samples underwent a quantitative analysis to monitor 17 known cyanotoxins  according to previously published method [Surface water sampling was conducted by the ATRAPP  research initiative co-financed by Genome Quebec and Genome Canada. The samples were collected in the photic zone of several lakes under surveillance, due to their occurrence of toxic algal blooms located in Canada, United Kingdom and France . At eachthe site . The botthe site . A volumd method . SamplesTM 3000 RS pump and column compartment were used for chromatographic separation. The Dionex UltiMateTM 3000 pump was coupled to the system used for on-line solid phase extraction (SPE), and both were controlled by Chromeleon 7.2 Software . A PAL system RTC autosampler was used  for injection. A Hypersil Gold  column was used for on-line SPE, and the chromatographic separation was done with a Hypersil Gold  column kept at 55 °C. Analysis of samples was performed using a Q-Exactive mass spectrometer controlled by the Xcalibur 3.0 software . Instrument calibration in positive mode was done every 7 days with a direct infusion of an LTQ Velos ESI Positive Ion Calibration Solution , i.e., a mixture of caffeine, Met-Arg-Phe-Ala (MRFA) and Ultramark 1621 to reach mass accuracy within the 5 ppm range. Mass accuracy for all target compounds remained in the 5 ppm range in the 7-days post calibration.A Thermo Scientific Dionex UltiMate-1. A washing volume of 0.5 mL passed through the column following the sample loading step. The pre-concentration columns were finally back-flushed with MeOH and the eluting analytes were transferred using the analytical pump gradient directly through the analytical column and chromatographic separation is proceeded with the solvents acetonitrile (B), and water (A) with the addition of 0.1% formic acid at a flow rate of 525 µL min-1. A total chromatographic run of 8 min was carried out for the first screening step of the samples . The chromatographic run was extended to 30 min to ensure better chromatographic separation when using the suspect screening method via DIA mode. These chromatographic parameters were also applied to the samples, including calibration curve and quality control standards for semi-quantification , an unknown compound grouping and features merging, and a blank subtraction using uncontaminated lake water samples. Then, the grouped compounds were investigated in the in-house database searching with a mass tolerance of 5 ppm and retention time tolerance of 0.05 min. These databases were individually constructed for MCs and APs in Excel® sheets, including the masses values from all the possible congener’s combinations minus the duplicates, which resulted in 8,709 individual masses for MCs and 8,815 individual masses for APs. Afterwards, a composition prediction was achieved, including minimum and maximum element counts , and the maximum includes all possible elements present in all congeners. Finally, only the compounds detected in duplicate with a coefficient of variation of the signal intensity lower than 30% were retained for later steps. The FS data were first processed using Compound Discoverer 3.0 . The workflow was built for the search of unknown compounds with in-house database searches, including all suspected cyanopeptides. MCs and APs were processed separately with the same workflow, but different database lists built according to the different molecular combinations, based on the potential amino acids in the molecules  5,7,10,,10,11,29m/z 163.11229-Adda-134-NH3+H+), and a second specific to the form of Adda (m/z 135.08099—Adda and (6Z)Adda, m/z 121.06534-DMAdda, m/z 163.07591-ADMAdda). For APs, the m/z 84.08136 fragment was used as a marker that corresponds to the immonium ion of lysine, an amino acid found in all APs. Though the mass is low, this fragment is rarely found in environmental samples when lysine is not present [The following data treatment was performed using the Xcalibur 3.0 Software . MCs and APs have both few distinctive fragments, which were used to narrow down the list of features, and these were searched in the MS/MS spectra from the DIA acquisition. For MCs, the Adda function was the specific marker with two fragments simultaneously found in a MS/MS spectra: A first common to all congeners ( present . Finally